Studies on the active site of bovine liver argininosuccinase have been initiated and will be continued. Bromo analogs of fumaric acid proved to be the most effective chemical modifying reagents. Enzyme inactivation by the reagent under mild conditions was completely prevented by the substrate, argininosuccinate. The specificity for alkylation at the active site indicated by substrate protection and the results of preliminary experiments on the stoichiometry of alkylation were both promising. Our proposed work will be directed toward the isolation of a peptide fragment uniquely modified by the alkylating agent and toward identification of the amino acid that has undergone modification. The expectation that after tryptic digestion only one peptide fragment should contain the modified amino acid is based on our previous studies showing that argininosuccinase of mol. wt. 202,000 is a tetramer composed of four identical subunits, each containing one catalytic site. Enzyme studies on bovine brain argininosuccinase have recently been resumed and it is planned to continue purification and study of the properties of the brain enzyme for comparative purposes. It is expected that comparison with the liver enzyme from the same species will establish whether the enzyme from two organs of the same species are identical proteins. The answer to this question carries considerable genetic and functional significance for general basic reasons and specifically for the reason that reported studies on humans afflicted with a mutation resulting in reduced activity of liver argininosuccinase contain conflicting results on the enzymatic activity in the kidney or brain of the same individuals. Both normal and decreased values have been reported. Bibliographic references: Determination of Argininosuccinate in Normal Blood Serum and Liver. S. Ratner, Analytical Biochemistry, 63, 141-155 (1975).